The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Comparative hybridization methods test the ability of two nucleic acids to interact with a third target nucleic acid. In particular, comparative genomic hybridization (CGH) is a method for detecting chromosomal abnormalities. CGH was originally developed to detect and identify the location of gain or loss of DNA sequences, such as deletions, duplications or amplifications commonly seen in tumors (Kallioniemi et al., Science 258:818-821, 1992). For example, genetic changes resulting in an abnormal number of one or more chromosomes (i.e., aneuploidy) have provided useful diagnostic indicators of human disease, specifically as cancer markers. Changes in chromosomal copy number are found in nearly all major human tumor types. For a review, see Mittelman et al., “Catalog of Chromosome Aberrations” in CANCER, Vol. 2 (Wiley-Liss, 1994).
In addition, the presence of aneuploid cells has also been used as a marker for genetic chromosomal abnormalities. Various chromosomal abnormalities may occur in an estimated 0.5% of all live births. For example, Down's syndrome or trisomy 18 which has an incidence of about 1 in 800 live births, is commonly the subject of a variety of prenatal screens or diagnostic techniques. Chromosomal aneuploidies involving chromosomes 13, 18, 21, X and Y account for up to 95% of all liveborn chromosomal aberrations resulting in birth defects (Whiteman et al., Am. J. Hum. Genet. 49:A127-129, 1991), and up to 67% of all chromosomal abnormalities, including balanced translocations (Klinger et al., Am. J. Hum. Genet. 51:52-65, 1992).
CGH is useful to discover and map the location of genomic sequences with variant copy number without prior knowledge of the sequences. Oligonucleotide probes directed to known mutations are not required for CGH. Early CGH techniques employ a competitive in situ hybridization between test DNA and normal reference DNA, each labeled with a different color, and a metaphase chromosomal spread. Chromosomal regions in the test DNA, which are at increased or decreased copy number as compared to the normal reference DNA can be quickly identified by detecting regions where the ratio of signal from the two different colors is altered. For example, those genomic regions that have been decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference (compared to other regions of the genome (e.g., a deletion)); while regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA (e.g., a duplication). Where a decrease or an increase in copy number is limited to the loss or gain of one copy of a sequence, CGH resolution is usually about 5-10 Megabases (Mb).
CGH has more recently been adapted to analyze individual genomic nucleic acid sequences rather than a metaphase chromosomal spread. Individual nucleic acid sequences are arrayed on a solid support, and the sequences can represent the entirety of one or more chromosomes, or the entire genome. The hybridization of the labeled nucleic acids to the array targets is detected using different labels, e.g., two color fluorescence. Thus, array-based CGH with a plurality of individual nucleic acid sequences allows one to gain more specific information than a chromosomal spread, is potentially more sensitive, and facilitates the analysis of samples.
For example, in a typical array-based CGH, equitable amounts of total genomic nucleic acid from cells of a test sample and a normal reference sample are labeled with two different colors of fluorescent dye and co-hybridized to an array of BACs, which contain the cloned nucleic acid fragments that collectively cover the cell's genome. The resulting co-hybridization produces a fluorescently labeled array, the coloration of which reflects the competitive hybridization of sequences in the test and reference genomic DNAs to the homologous sequences within the arrayed BACs. Theoretically, the copy number ratio of homologous sequences in the test and reference genomic nucleic acid samples should be directly proportional to the ratio of their respective colored fluorescent signal intensities at discrete BACs within the array. Array-based CGH is described in U.S. Pat. Nos. 5,830,645 and 6,562,565 for example, using target nucleic acids immobilized on a solid support in lieu of a metaphase chromosomal spread.
When combining more than one color or type of labeled nucleic acid in a hybridization mixture, the relative concentrations and/or labeling densities may be adjusted for various purposes. Adjustments may be made by selecting appropriate detection reagents (avidin, antibodies and the like), or by the design of the microscope filters among other parameters. When using quantitative image analysis, mathematical normalization can be used to compensate for general differences in the staining intensities of different colors. Thus, the use of different labels to distinguish test from reference genomic nucleic acids in traditional CGH entails additional refinements or adjustments that complicate sample processing, standardization across samples, and evaluation of the results obtained. For example, when using visual observation or photography of the results, the individual color intensities need to be adjusted for optimum observability of changes in their relative intensities.
Although CGH is a powerful tool for genetic analysis, CGH has not been used to detect balanced chromosomal translocations. A chromosomal translocation is a type of genetic anomaly that occurs when genetic material from one chromosomal region transfers to another. The phenotypic effects of certain translocations may be minor or unnoticeable, however, some translocations may have more severe phenotypic consequences including mental retardation, infertility, congenital malformations, and dysmorphic features.
Reciprocal and Robertsonian translocations are the most frequently occurring types of translocations. Reciprocal translocations usually involve a two-way exchange between different chromosomes. The chromosomes break apart and segments below the break points swap positions. If the event is balanced, no net gain or loss of genetic material results and the individual is usually phenotypically unaffected if no genes are disrupted. However, gametes of that individual have the potential to be unbalanced with an excess of certain regions and/or an absence of others. This creates the possibility for both balanced and unbalanced progeny.
A translocation of genetic material between human chromosomes 9 and 22, commonly known as the Philadelphia chromosome, is one of the most extensively studied balanced translocation occurrences. This exchange causes most of the proto-oncogene ab1 normally on chromosome 9 to translocation to a break point chromosome 22 which occurs in the middle of the bcr gene of that chromosome. Thus, the translocation results in an expressible fused gene BCR-Ab1 that includes the 5′ portion of the bcr gene and much of the ab1 gene. The improper ab1 gene function of the Philadelphia chromosome is associated with Chronic Myelogenous Leukemia (CML).
In other instances, Sotos Syndrome can occur from balanced or unbalanced translocations that cause a disruption of the NDS1 gene. This disease is characterized by large body and head size with distinct facial features.
Robertsonian translocations occur when two chromosomes fuse at the centers and essentially combine into one. Most of the genetic material remains from both chromosomes. As in balanced reciprocal translocations, the carrier may be normal, but produce genetically unbalanced gametes. Most progeny originating from unbalanced gametes do not survive and a miscarriage occurs during early pregnancy. If the carrier is fertile and progeny survive, various defects could occur. One Robertsonian translocation results in the fusion of chromosomes 14 and 21. Resulting progeny may inherit three copies of chromosome 21 which causes Down's syndrome.
A variety of methods have been used to detect balanced translocations. One of the most frequently employed methods is fluorescent in situ hybridization (FISH). Fluorescent probes detect and physically map regions of interest within chromosomes, cells, or tissues on a microscope slide. FISH is relatively quick method sensitive to minor exchanges and has a resolution minimum of about 10 Mb. In FISH, a fluorescent probe hybridizes to the sample and is visually detected. Other methods frequently used to detect balanced translocations include karyotyping, flow cytometry, and DNA microarray analysis in conjunction with physical mapping. Karyotyping is an actual picture of the chromosomes of a cell. Flow cytometry identifies cell changes by laser identified tags for an antibody for antigens known to be present in malignant cells.
Previous CGH methods were unable to detect balanced translocations because the previous CGH methods rely on the detection of relative differences between test and reference samples whereas balanced translocations result in equal chromosomal exchange thereby maintaining the same relative quantities. The present invention provides methods in which CGH may be used to detect balanced translocations.